Thyrotropin releasing hormone (TRH) occurs naturally in humans and is recognized to have multifaceted, homeostatic, neuroprotective and neurotrophic effects in the central nervous system (CNS), which are Independent of its endocrine actions. These CNS effects of TRH provide the basis for its clinical use in the treatment of CNS-related disorders and may confer significant advantages for TRH-based compounds over other prospective neurotherapeutics directed towards a single injury mechanism, particularly in complex CNS disease states, such as neurodegenerative disorders. Potential clinical applications of TRH and TRH-related compounds in human CNS-related disorders include, but are not limited to, depression, chronic fatigue syndromes, narcolepsy, neurasthenia, lethargy, sedation secondary to drugs, chemo- or radiation therapy, sedative intoxication/respiratory distress, recovery from general anaesthesia, attention deficit/hyperactive disorder (ADHD), disturbances of circadian rhythm (e.g. jet lag), bipolar affective disorder, anxiety disorders, Alzheimer's disease and other dementias with cognition deficits, frontotemporal lobe dementia, seizure disorders, obesity and motor neuron disorders and pain. Clinical use of TRH is limited, however, by its short half life and potential endocrine side effects. U.S. Pat. No. 7,713,935 B2 describes how Glp-Asn-Pro-DTyr-DTrp-NH2 overcomes these drawbacks and offers a means to harness therapeutic benefits of central TRH actions in the treatment of a wide range of CNS disorders.
The biological actions of neuroactive peptides, such as naturally-occurring TRH, are mediated by specific receptors. Ligand-receptor interaction can be measured by radioligand binding studies or less directly through assessment of dose-response curves for a biological effect. The former requires a radiolabelled form of the ligand and a source of receptors, such as a cell membrane fraction, intact cells or solubilised preparations. The receptors and ligand are incubated together until equilibrium is reached and the amount of labelled ligand bound to the receptors is determined. When homogenised tissue/particulate preparations are used, this may be accomplished by filtration, centrifugation or equilibrium dialysis which separate free ligand from receptor-bound ligand. With soluble tissue preparations this separation of bound from free may be achieved by gel filtration, equilibrium dialysis, and/or precipitation of the receptor-bound ligand. Ligand binding screening assays are useful to identify new compounds that target a receptor. Competition binding assays, in which an unlabelled test compound is tested for its ability to displace the radiolabelled ligand, can be used to determine the affinity of such other compounds for the receptor site. Autoradiography employing radiolabelled ligand can be used to localize and map regional receptor distribution, estimate the CNS uptake of ligand, and carry out pharmacokinetic evaluations.
[3H][3-Me-His2]TRH is typically employed to label high affinity TRH receptor sites in radioligand binding studies since it binds with greater affinity and affords higher specific binding than [3H]TRH.
A limited number of radioligand binding studies employing human brain tissue have been described. These studies have shown that [3H][3-Me-His2]TRH-labelled receptors are present in discrete areas in the human brain with highest levels of binding found in the limbic structures. In human amygdala, binding of [3H][3-Me-His2]TRH was observed to be saturable and displayed a Kd of 7-10 nM. [3H][3-Me-His2]TRH bound to human pituitary with similar affinity, though fewer binding site were observed in this tissue compared to the amygdala.
Investigation of [3H]TRH and [3H][3-Me-His2]TRH binding in rodent brain tissue has been more extensively reported. Data from such studies show that specific [3H][3-Me-His2]TRH binding sites are distinctly localised, and indicate that this radiolabelled peptide binds to a single population of high affinity sites on rat brain cortical membranes with a Kd of around 5 nM and that TRH competes for these sites with a Ki value of around 25 nM. Similarly, [3H][3-Me-His2]TRH appears to bind to a single population of high-affinity sites in rat pituitary tissue with a Kd of 2.2 nM.
G-protein-coupled receptors (GPCRs) are recognized to be involved in mediating the biological actions of neuropeptides and are viewed as attractive neuropharmacological targets. To date, two GPCR subtypes for TRH have been identified in non-primates: TRH receptor 1 (TRHR1) and TRH receptor 2 (TRHR2). In addition, a third putative TRH receptor subtype was cloned in Xenopus laevis (xTRHR3); however, because xTRHR3 exhibited very low affinity for TRH and TRH analogs and did not discriminate among the analogs, the authors subsequently suggested that xTRHR3 is likely a receptor for another peptide.
Comparison of amino acid sequences of TRHR1 and TRHR2 from the same species shows that they have an overall homology of around 50%. In the rat, the distribution patterns of TRHR1 and TRHR2 are quite distinct. For example, TRHR1 is expressed at high levels in the pituitary and displays limited expression in the central nervous system (CNS), whereas TRHR2 is absent or present only at low levels in the pituitary and is widely distributed throughout the CNS. The distinct regional distribution of the mRNAs for TRHR1 and TRHR2, has led to the notion that TRHR1 plays a principal role in mediating the endocrine functions of TRH, while TRHR2 may be important in mediating the higher cognitive functions of TRH, as well as its effects on arousal, locomotor activity and pain perception.
Prior to cloning studies identifying two receptor subtypes for TRH in rodents it had been shown that TRH receptor protein isolated from rat brain had an isoelectric point (i.e. PI=5.5) that differed from that isolated from rat pituitary (i.e. PI=4.9), indicating that the TRH receptors in rat brain could be structurally different from those in rat pituitary.
Both TRHR1 and TRHR2 display similar high affinity for [3H][3-Me-His2]TRH and so this ligand cannot be used to discriminate between these two known TRH receptor subtypes. Similarly, TRH and many TRH analogs fail to discriminate pharmacologically between these two TRH receptor subtypes. Nevertheless, a few compounds have been reported that appear to display a degree of selectively for binding to TRHR2 versus TRHR1 in cells expressing either TRHR1 or TRHR2. Glp-Asn-Pro-DTyr-DTrp-NH2 does not displace [3H][3-Me-His2]TRH binding from cells expressing either TRHR1 or TRHR2, or rat pituitary tissue homogenate; however, it does displace [3H][3-Me-His2]TRH binding in native rat cortical tissue (Scalabrino et al., 2007).
With the discovery of TRHR2 in rodents it was initially thought that this may provide a therapeutic target for developing TRH-based neurotherapeutics for use in humans; however, TRHR2 has not been found to be present in humans. U.S. Pat. No. 6,441,133 discloses the structure of TRHR2 and claims pure protein comprising the amino acid sequence identified for TRHR2, as well as isolated recombinant TRHR2. In addition, this patent is directed to a method for assaying a test compound for its ability to bind to TRHR-2 and for assaying a test compound for its ability to alter the expression of the TRHR-2 gene. An earlier patent—U.S. Pat. No. 5,288,621—disclosed for the first time is the isolation, sequence, and expression cloning of a cDNA encoding for pituitary TRHR (i.e. TRHR1), as well as the amino acid sequence for this receptor.
Human TRHR1 is approximately 90% homologous with mouse and rat TRHR1 at the cDNA and amino acid level.
Notably, TRHR2 is not detected in humans; the only TRH receptor that has been cloned in humans is the TRHR1 subtype. Thus, to date the art indicates that in humans there is only one homogeneous TRH receptor subtype. The art also indicates that TRH receptors in human brain and pituitary are indistinguishable.
Competitive radioligand binding studies provide an important means to enable the discovery of new receptor subtypes, as well as pharmacological characterisation and classification of receptor subtypes. Receptor subtypes may be defined pharmacologically. In such cases, subtypes may be distinguished from one another on the basis of differential binding of different ligands.
The use of animal tissues and heterologous cells expressing a particular receptor subtype in drug development has its drawbacks, as there can be differences in receptor subtypes between humans and animals, such that activity in animals may not translate into efficacy in humans. Importantly, the mediation of ligand signalling through GPCRs was initially understood to involve monomeric receptors. However, this view has been revised recently with the recognition that these receptors form homo-oligomeric and hetero-oligomeric complexes that influence GPCR receptor functioning and have implications regarding drug design. For example, pairings of μ and δ subtypes of opioid receptors result in reduced affinity for ligands that are specific for each subtype. Also in relation to this, it has been suggested that data gathered from studies using isolated receptors in a non-physiological state may be misleading since the possibility of GPCR homo-hetero oligomerisation, which may be essential for ligand-receptor interactions and/or signaling, may not be possible under such circumstances. In the case of TRH receptors, constitutive and agonist-induced homo-oligomerisation has been demonstrated, as well as TRH receptor subtype hetero-oligomer formation. Thus, it is possible that formation of TRH receptor heterocomplexes may occur in native tissue, which would not be possible in the cell models expressing a single receptor subtype.
Confirmation of the binding of a potential drug to native human receptors using radioligand competition binding assays is increasingly recognised to be an important step in preclinical drug development.
It has previously been shown that Glp-Asn-Pro-D-Tyr-D-TrpNH2 binds with high affinity to native TRH receptors labelled with [3H][3-Me-His2]TRH in rat cortical and hippocampal tissue homogenates; although it does not displace [3H][3-Me-His2]TRH binding from native rat pituitary tissue, CHO-TRHR1, CHO-TRHR2 or GH4 membranes (Scalabrino 2007, Hogan 2008). Glp-Asn-Pro-LTyr-LTrp-LTrp-AMC, Glp-Asn-Pro-DTyr-DTrp-DTrpAMC, Glp-Asn-Pro-LTyr-LTrp-AMC, Glp-Asn-Pro-DTyr-DTrpAMC, Glp-Asn-Pro-D-Tyr-D-TrpNH2, Glp-Asn-Pro-LTyr-LTrp-LTrpNH2, and Glp-Asn-Pro-LTyr-LTrp-NH2, display these same discriminatory properties as Glp-His-Pro-D-Tyr-D-TrpNH2. Thus, this family of peptides does not displace [3H][3-Me-His2]TRH binding in a GH4 pituitary cell line, which naturally expresses TRHR1; however, these peptides do displace [3H][3-Me-His2]TRH binding in native rat brain cortical and hippocampal and display high affinity (i.e. Ki values <10−6 M) for these binding sites in these tissues. In contrast, [3-Me-His2]TRH displays high affinity for [3H][3-Me-His2]TRH-labelled sites in both GH4 and native rat brain cortical and hippocampal tissues (see Table 1). This family of peptides has previously been described in U.S. Pat. Nos. 7,378,397 B2 and 7,713,935 B2 as novel chemical entities that inhibit the TRH-degrading ectoenzyme (TRH-DE).
The present invention shows that Glp-Asn-Pro-D-Tyr-D-TrpNH2 does not displace [3H][3-Me-His2]TRH binding from human pituitary tissue. Unexpectedly, however, given that research indicates the presence of only the TRHR1 subtype in humans, Glp-Asn-Pro-D-Tyr-D-TrpNH2 was found to displace [3H][3-Me-His2]TRH binding from CNS tissue. This finding demonstrates for the first time that a TRH analog i.e. Glp-Asn-Pro-D-Tyr-D-TrpNH2, binds selectively, with nM affinity, to a novel TRH receptor subtype in human CNS tissue that is pharmacologically distinct from the TRH receptor in human pituitary tissue. Notably, Glp-Asn-Pro-D-Tyr-D-TrpNH2 provides a groundbreaking innovative tool to distinguish between these two, hitherto unrecognised, pharmacologically-distinct human TRH receptor subtypes.
Thus, Glp-Asn-Pro-DTyr-DTrp-NH2 and the family of structurally-related compounds defined in the claims, including Glp-Asn-Pro-LTyr-LTrp-LTrp-AMC, Glp-Asn-Pro-DTyr-DTrp-DTrpAMC, Glp-Asn-Pro-LTyr-LTrp-AMC, Glp-Asn-Pro-DTyr-DTrpAMC, Glp-Asn-Pro-D-Tyr-D-TrpNH2, Glp-Asn-Pro-LTyr-LTrp-LTrpNH2, and Glp-Asn-Pro-LTyr-LTrp-NH2, provide a unique means to recognise the existence of this novel TRH receptor subtype—no other compounds had been previously identified that can discriminate between this new central TRH receptor and the TRH pituitary receptor. Hence, there are no existing solutions to understanding how the biological effects of TRH in the CNS are mediated.
U.S. Pat. No. 5,879,896 principally claims a method of screening for a compound that inhibits binding of TRH to a human TRH receptor, or a salt thereof, comprising contacting a TRH receptor protein obtained from a cell transformed with an expression vector containing a DNA encoding a TRH receptor having the amino acid sequence of TRHR1, or a sufficient portion thereof to bind TRH, or the salt thereof, with the compound to be screened and TRH, and comparing binding between TRH and the TRH receptor in the absence and presence of the compound, wherein less binding between the TRH and the receptor in the presence of the compound than in the absence of the compound is indicative of the compound inhibiting binding between TRH and the receptor. Clearly, since Glp-Asn-Pro-DTyr-DTrp-NH2 and the structurally-related family of peptides described above were not discovered until the 2000s, the inventors of U.S. Pat. No. 5,879,896 could not have possibly anticipated the existence of a TRHR subtype that is revealed by Glp-Asn-Pro-DTyr-DTrp-NH2 binding not the use of this and related compounds as described herein.
The invention described herein is relevant to the development of diagnostics and therapeutics for any TRH-related disorders, inter alia, brain and spinal injury, memory loss, spinocerebellar degeneration, pain including spinal cord pain, epilepsy, eating disorders, weight management disorders (particularly obesity), and CNS-related diseases, as well as memory loss, lethargy, anxiety disorders, jet lag, attention deficit disorders, post-traumatic syndrome and as a mood stabilizer or enhancer, and may also have application as a research tool to investigate TRH-mediated cellular processes. The present invention opens up a new area of study for pharmacological intervention of TRH signalling in the CNS and has important implications for the treatment of and development of therapeutics for CNS disorders.
The invention described herein provides for the first time a means to understand how the central therapeutic effects of TRH are mediated, as well as a method for screening for compounds that interact with this novel TRHR site that can be pharmacologically distinguished by Glp-Asn-Pro-DTyr-DTrp-NH2 and the family of structurally-related compounds defined herein.